High viscosity xanthan polymer preparations

ABSTRACT

Increasing the molecular length of xanthan polymer makes a higher viscosity xanthan composition. Xanthan with higher specific viscosity characteristics provides more viscosity at equivalent concentration in food, industrial and oilfield applications. Methods for increasing the viscosity of xanthan include inducing particular key genes and increasing copy number of particular key genes.

[0001] This application claims the benefit of provisional applicationU.S. Ser. No. 60/456,245 filed Mar. 21, 2003.

[0002] A portion of the disclosure of this patent document containsmaterial which is subject to copyright protection. The copyright ownerhas no objection to the facsimile reproduction by anyone of the patentdocument or the patent disclosure, as it appears in the Patent andTrademark Office patent file or records, but otherwise reserves allcopyright rights whatsoever.

FIELD OF THE INVENTION

[0003] The invention relates to the field of microbial products. Inparticular it relates to microbial products having improved propertiesfor various industrial purposes.

BACKGROUND OF THE INVENTION

[0004] The chemical structure of xanthan is composed of a linearcellulosic (1→4)-β-D-glucose polymer with trisaccharide side chainscomposed of mannose, glucuronic acid and mannose, attached to alternateglucose residue in the backbone. (Milas and Rinaudo, CarbohydrateResearch, 76, 189-196, 1979). Thus xanthan can be described as abranched chain polymer with a pentasaccharide repeat unit; normalxanthan typically has 2000-3000 pentasaccharide repeat units. Thexanthan polymer is typically modified by acetylation and pyruvylation ofthe mannose residues.

[0005] The fermentation of carbohydrates to produce the biosyntheticwater-soluble polysaccharide xanthan gumBy the action of Xanthomonasbacteria is well known. The earliest work was conducted by the UnitedStates Department of Agriculture and is described in U.S. Pat. No.3,000,790. Xanthomonas hydrophilic colloid (“xanthan”) is an exocellularheteropolysaccharide.

[0006] Xanthan is produced by aerobic submerged fermentation of abacterium of the genus Xanthomonas. The fermentation medium typicallycontains carbohydrate (such as sugar), trace elements and othernutrients. Once fermentation is complete, the resulting fermentationbroth (solution) is typically heat-treated. It is well established thatheat treatment of xanthan fermentation broths and solutions leads to aconformational change of native xanthan at or above a transitiontemperature (TM) to produce a higher viscosity xanthan. Heat treatmentalso has the beneficial effect of destroying viable microorganisms andundesired enzyme activities in the xanthan. Following heat-treatment,the xanthan is recovered by alcohol precipitation. However, heattreatment of xanthan fermentation broths also has disadvantages, such asthermal degradation of the xanthan. Heating xanthan solutions or brothsbeyond TM or holding them at temperatures above TM for more than a fewseconds leads to thermal degradation of the xanthan. Degradation ofxanthan irreversibly reduces its viscosity. Accordingly, heat treatmentis an important technique with which to control the quality andconsistency of xanthan.

[0007] Xanthan quality is primarily determined by two viscosity tests:the Low Shear Rate Viscosity (“LSRV”) in tap water solutions and the SeaWater Viscosity (“SWV”) in high salt solutions. Pasteurization ofxanthan fermentation broths at temperatures at or above TM has beenfound to yield xanthan of a higher viscosity as indicated by higher LSRVand SWV values.

[0008] Xanthan polymer is used in many contexts. Xanthan has a widevariety of industrial applications including use in oil well drillingmuds, as a viscosity control additive in secondary recovery of petroleumby water flooding, as a thickener in foods, as a stabilizing agent, andas a emulsifying, suspending and sizing agent (Encyclopedia of PolymerScience and Engineering, 2nd Edition, Editors John Wiley & Sons,901-918, 1989). Xanthan can also be used in cosmetic preparations,pharmaceutical vehicles and similar compositions.

[0009] There is a need in the art to produce a xanthan polymer withhigher specific viscosity characteristics in the unpasteurized state.Such a higher specific viscosity xanthan polymer could provide moreviscosity at equivalent xanthan concentrations, for example, for food,industrial, and oilfield applications.

BRIEF SUMMARY OF THE INVENTION

[0010] In a first embodiment an unpasteurized xanthan composition isprovided. The composition can be provided by a cell which over-expressesgumB and gumC. It has an intrinsic viscosity which is at least 20%greater than xanthan from a corresponding strain which does notover-express gumB and gumC.

[0011] In a second embodiment a xanthan composition is provided. Itcomprises a population of xanthan molecules having a range of molecularlengths. At least 1% of the population has a length greater than 3 um asmeasured by atomic force microscopy.

[0012] In a third embodiment of the invention a method is provided forproducing a xanthan polymer preparation having increased viscosityrelative to that produced by a wild-type strain. The amount of geneproduct of gumB and gumC is selectively increased in a Xanthomonascampestris culture. The amount of a gene product of orfX is notselectively increased. Nor is the amount of a product of a gene selectedfrom the group consisting of gumD-gumG selectively increased. A higherviscosity xanthan polymer preparation is thereby produced by theculture.

[0013] In a fourth embodiment of the invention a method is provided forproducing a xanthan polymer preparation having increased viscosityrelative to that produced by a wild-type strain. A Xanthomonascampestris strain is cultured in a culture medium under conditions inwhich it produces a xanthan polymer. The strain selectively producesrelative to a wild-type strain more gene product of gumB and gumC butnot of orfX nor of a gene selected from the group consisting ofgumD-gumG.

[0014] In a fifth embodiment of the invention an unpasteurized xanthancomposition is provided. The composition is made by a cell whichover-expresses gumB and gumC. The composition has a seawater viscositywhich is at least 10% greater than xanthan from a corresponding strainwhich does not over-express gumB and gumC.

[0015] The present invention thus provides the art with xanthancompositions which have increased viscosity relative to those similarlyproduced by corresponding wild-type strains.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows genetic constructs relative to a genetic map of thegumB-M operon, also known as the xpsB-M (xanthan polysaccharidesynthesis) operon.

[0017]FIGS. 2A and 2B show Western blot analyses of gumB and gumCprotein product expression, respectively.

[0018]FIG. 3 shows an intrinsic viscosity plot for xanthan gum samples,one of which over-expresses gumB and gumC gene products due to thepresence of a plasmid carrying extra copies of the genes.

DETAILED DESCRIPTION OF THE INVENTION

[0019] It is a discovery of the present inventors that overexpression ofgumB and gumC gene products relative to other genes in their operon,yields xanthan products with higher viscosity on a per weight basis.While applicants do not wish to be bound by any particular theory ofoperation, it appears that a shift in the ratio of certain gene productsleads to a shift in the size distribution of xanthan polymer molecules.A significant number of molecules are of higher molecular length thanwhen xanthan is made by a wild-type cell. These longer molecules lead toa higher viscosity of the population or preparation.

[0020] It is known in the art that increases in viscosity can beobtained by pasteurizing xanthan preparations. See Talashek et al., U.S.Pat. No. 6,391,596. However, the increased viscosity found as the resultof overexpression of gumB and gumC is observed even in the absence ofpasteurization. Nonetheless subsequent pasteurization of the products ofthe present invention will yield an even more viscous preparation.

[0021] Overexpression of both gumB and gumC appear to be required toachieve the increased viscosity. When either gene was tested alone, theincrease was not observed. The overexpression of gumB and gumC can beassessed relative to other genes of the gumB-M operon. Whileoverexpression relative to any of those genes may be sufficient toachieve the effect, overexpression with respect to orfX and gumD may beparticularly significant. OrfX is a small open reading frame that waspreviously published as a segment of the genome designated as gumA,immediately upstream of gumB. Recently two open reading frames have beendiscerned in the former gumA region, ihf and orfX Overexpressionrelative to all of the genes gumD-gumM may be desirable.

[0022] Overexpression of the desired gene products may be achieved byany means known in the art, including, but not limited to, introducingadditional copies of the genes encoding the desired gene products to aXanthomonas campestris cell or other bacterium that makes xanthan, andinduction of the desired gene products using for example an induciblepromoter. Other bacteria that make xanthan include those that have beengenetically engineered to contain the xanthan biosynthetic genes. ThegumB and gumC genes can be introduced on one or more vectors, i.e., incombination or individually.

[0023] Inducible promoters which can be used according to the inventioninclude any that are known in the art, including the lac promoter, theara promoter, the tet promoter, and the tac promoter. Natural andartificial inducing agents for these promoters are known in the art, andany can be used as is convenient. Additional copies of genes can beintroduced on plasmids or viral vectors, for example. Additional copiesof the desired genes can be maintained extrachromosomally or can beintegrated into the genome.

[0024] Recovery of xanthan from a culture broth typically involves oneor more processing steps. The xanthan may be heat-treated. The xanthanmay be precipitated with an alchohol, such as isopropyl alcohol, ethylalcohol, or propyl alcohol. Typically the cells are not specificallyremoved from the culture broth.

[0025] Xanthan molecules produced biosynthetically typically have adistribution of sizes. The increased viscosity of the present inventionmay be achieved by increasing the number of molecules having a muchlonger than average length, or by increasing to a greater degree thenumber of molecules having a somewhat longer than average length. Thenumber of molecules which have increased length need not be huge. Atleast 1, 3, 5, 7, 9, or 11% of the molecules with an increased lengthmay be sufficient. The molecules of increased length may be greater than3, 4, 5, 6, 7, 8, or 9 um, as measured by atomic force microscopy. Thepercentage of the mass of the total xanthan population contributed bythe molecules which are longer than 3, 4, 5, 6, 7, 8, or 9 um will begreater than their number proportion in the population. Thus at least 1,3, 5, 10, 15, 20, or 25% of the total mass of the xanthan molecules maybe contributed by molecules having a greater than 3 um length.

[0026] Intrinsic viscosity measurements are yet another way tocharacterize the preparations of the present invention. Increases seenusing this type of measurement may be as great as 5, 10, 15, 20, 25, 30,or 35% over that produced by wild-type strains. Proper controls forcomparison purposes are those corresponding strains which are mostclosely related to the strains being tested. Thus if testing strainsthat have additional copies of gumB and gumC, the best control will havethe same genetic complement but for the presence of the additionalcopies of gumB and gumC. If testing cultures that have been induced byan inducer to produce more gumB and gumC gene product, then the bestcontrol will be cultures of the same strain that have not been induced.Sea water viscosity can also be used to characterize preparations of thepresent invention. Increases seen using this type of measurement may beas great as 5, 10, 15, 20, 25, 30, or 35% over that produced bywild-type strains.

[0027] Xanthan is used as a component in a number of products to improveproperties. The properties may include viscosity, suspension ofparticulates, mouth feel, bulk, to name just a few. Other propertiesinclude water-binding, thickener, emulsion stabilizing, foam enhancing,and sheer-thinning. Such products include foods, such as saladdressings, syrups, juice drinks, and frozen desserts. Such products alsoinclude printing dyes, oil drilling fluids, ceramic glazes, andpharmaceutical compositions. In the latter case, xanthan can be used asa carrier or as a controlled release matrix. Other products wherexanthan can be used include cleaning liquids, paint and ink, wallpaperadhesives, pesticides, toothpastes, and enzyme and cell immobilizers.

[0028] While the invention has been described with respect to specificexamples including presently preferred modes of carrying out theinvention, those skilled in the art will appreciate that there arenumerous variations and permutations of the above described systems andtechniques that fall within the spirit and scope of the invention as setforth in the appended claims.

EXAMPLES Example 1 Strain Construction

[0029] To isolate a fragment carrying the complete gum gene region of X.campestris, a genomic library of the wild type X. campestris strain,NRRL B-1459 (1), was constructed with the broad-host-range cosmid vectorpRK311 (2) by cloning of total DNA partially digested with Sau3AI. Thislibrary was mated en masse from E. coli S17-1 (3) to the Gum⁻ X.campestris mutant 2895 (4). One of the cosmids isolated from severalmucoid exconjugants termed pIZD15-261 (5) contains a 16-kb fragmentencompassing the complete gum region. See FIG. 1 for a graphicrepresentation and Table 1 for a listing of the genes of the operon.TABLE 1 List of genes designations in the chromosomal region encodingxanthan polysaccharide synthesis X. campestris ATCC13951 X. campestris(NRRL B- pv. campestris Chromosomal 1459) ATCC33913 Location* Functioninf himA (XCC2457) 2918744-2918448 integration host factor, alpha chainorfX (XCC2456) 2918464-2918111 transcriptional regulator xpsB gumB(XCC2454) 2917444-2916806 xanthan export xpsC gumC (XCC2453)2916731-2915385 xanthan export xpsD gumD 2915139-2913688 glucosyltransferase (XCC2452) xpsE gumE (XCC2451) 2913602-2912307 xanthanpolymerization xpsF gumF (XCC2450) 2912307-2911216 acetyl transferasexpsG gumG (XCC2449) 2911216-2910149 acetyl transferase xpsH gumH2910078-2908939 mannosyl transferase (XCC2448) xpsI gumI (XCC2447)2908939-2907893 mannosyl transferase xpsJ gumJ (XCC2446) 2907893-2906397xanthan export xpsK gumK (XCC2445) 2906014-2905130 glucuronictransferase xpsL gumL (XCC2444) 2905086-2904295 pyruvyl transferase xpsMgumM 2904284-2903496 glucosyl transferase (XCC2443) orf165 (XCC2442)2903458-2902964 unknown conserved hypothetical

[0030] For the construction of the pBBR5-BC plasmid, a 4026 bp fragmentfrom pIZD15-261 digested with SpeI-BglII was cloned between the XbaI andBamHI sites of pKmob19 (8), giving rise to pGum02-19S (5). A 2855 bpfragment was released from plasmid pGum02-19S by digestion with SphI.This fragment was cloned into pUC 18 (9), which was previously digestedwith SphI, forming pUC18-BCAS.

[0031] The final plasmid (pBBR5-BC) was constructed by cloning theHindIII-XbaI fragment, containing the gum promoter and gumB and gumCgenes, into HindIII-XbaI digested pBBR1-MCS5 (10) (GenBank accession no.U25061).

[0032] The nucleotide sequence of the resulting pBBR5-BC plasmid isshown in SEQ ID NO: 1. (The predicted amino acid sequences of gumB andgumC are shown in SEQ ID NOs: 2 and 3, respectively. Thisbroad-host-range, medium-copy-number plasmid is 7.6 kb in length and iscompatible with IncP, IncQ and IncW group plasmids, as well as withColE1- and P15a-based replicons. The presence of an origin of transfer(mobRK2) enables its transference by conjugation into a wide range ofbacteria when the RK2 transfer functions are provided in trans. It alsocarries the gentamicin resistance gene and it contains the pBluescriptII KS multiple cloning site located within the gene encoding the LacZ apeptide (pBluescript II KS from Stratagene, La Jolla, Calif., USA).

[0033] To verify the expression of GumB and GumC proteins from pBBR5-BC,the plasmid was introduced into X. campestris mutant 1231, in which theentire gum (xps) gene cluster was deleted. Both proteins were detectedby Western blot in the mutant strain. TABLE 2 Bacterial strains andplasmids used or constructed in this work. Source Bacterial strain orplasmid Relevant characteristics (reference) E. coli. DH5α F - endA1hsdR17 supE44 thi-1 recA1 gyrA relA1 ΔlacU169 New England (φ80dlacZΔM15)Biolabs S17-1 E. coli 294 RP4-2-Tc::Mu-Km::Tn7 (3) JM109 F′ traD36proA⁺B⁺ lacl^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV44 e14 New England gyrA96ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal [dcm] [lon] Biolabs BL2I(DE3) F -ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal [dcm] [lon] (DE3) Novagen X.campestris NRRL B-1459 Wild type. (1) 2895 Rif^(r) xpsI-261 (11) 1231Tc::Tn 10 ΔxpsI C.P. Kelco XWCM1 Mutant of NRRL B-1459 C.P. Kelco PRM-1Mutant of NRRL B-1459 C.P. Kelco Plasmids pRK311 oriV(RK2) Tc^(r)oriT(mob⁺) tra⁻ λcos lacZ(α) (2) pIZD15-261 Cosmid based on pRK311carrying the X. campestris gum (5) region. pK19mob Km^(r), pK19derivative, mob-site (8) pgum02-19AS pK19mob vector carrying the gumfragment 770-4795^(a) (5) pUC18 Ap^(r), Co1E1, lacZα⁺ (9) pUC18-BCASpUC18 vector carrying the gum fragment 770-3610^(a) This work pBBR1-MCS5Gm^(r), pBBR1CM derivative, mob-site, lacZα⁺ (10) pBBR5-BC PBBR1-MCS5carrying the gum fragment 770-3610^(a) This work pQE-Xps#6 pQE30 vectorcarrying the gum fragment 1336-1971^(a) C.P. Kelco pQE30 Ap^(r) QiagenpREP4 Km^(r) Qiagen pET-C pET22b(+) vector carrying the gum fragment2135-3319^(a) This work pET22b+ Ap^(r) Novagen pH336 pRK290 carrying gumBamHI fragments1-15052^(a) Synergen pCOS6 pRK293 carrying Sa1I fragments1-14585a and upstream xps I DNA CP Kelco pFD5 pRK404 carrying partialBamHI gum fragment 318-3464^(a) Ielpi pCHC22 pRK293 carrying Sa1Ifragments 1-9223a and upstream xps I DNA (4) pBBR-prom pBBR1-MCS5carrying gum fragment 1000-1276^(a) This work pBBR5-B pBBR1-MCS5carrying gum fragment 770-1979^(a) This work pBBR-promC pBBR1-MCS5carrying gum fragment 1979-3459^(a) This work

[0034] Bacterial strains, plasmids, and growth conditions. The strainsand plasmids used in this study are listed in Table 2. E. coli strainswere grown in Luria-Bertani medium at 37° C. X. campestris strains weregrown in TY (5 g of tryptone, 3 g of yeast extract, and 0.7 g of CaCl₂per liter of H₂O) or in YM medium (12) at 28° C. Antibiotics from Sigma(St. Louis, Mo.) were supplemented as required at the followingconcentrations (in micrograms per milliliter): for X. campestris,gentamicin, 30; and tetracycline, 10; for E. coli, gentamicin, 10;kanamycin, 30; ampicillin, 100; and tetracycline, 10.

[0035] DNA biochemistry. Plasmid DNA from E. coli and X. campestris wasprepared by using the QIAprep Spin Miniprep Kit (QIAGEN, Hilden,Germany). DNA restriction, agarose gel electrophoresis and cloningprocedures were carried out in accordance with established protocols(13). All constructs were verified by DNA sequencing. Plasmid DNA wasintroduced into E. coli and X. campestris cells by electroporation asinstructed by Bio-Rad (Richmond, Calif.) (used parameters: E. coli: 200Ω, 25 μF, 2500V and X. campestris: 1000 Ω, 25μF, 2500V).

[0036] Analysis of nucleotide and protein sequences. The nucleotide andamino acid sequences were analyzed by using the MacVector SequenceAnalysis Software (Oxford Molecular Limited, Cambridge, UK).

Example 2 Western Analysis of gumB and gumC Expression

[0037] Western Analysis confirmed that gumB and gumC gene products arebeing over-expressed in the X. campestris strain with extra copies ofgumB and gumC. See FIG. 2.

Example 3 Intrinsic Viscosity determination

[0038] Xanthan samples prepared from X. campestris strains with(XWCM1/pBBR5BC) and without (XWCM1) multiple, plasmid encoded copies ofthe gumB and gumC genes were compared. Shake flask fermentations, usingglucose as a carbon source, were carried out to obtain xanthan fromthese strains.

[0039] Intrinsic viscosity was determined by measuring viscosity on bothpurified and unpurified xanthan samples. An increase in the intrinsicviscosity for xanthan from X. campestris strain with multiple copies ofgumB and gumC was observed. Intrinsic viscosity is proportional to themolecular weight for a given polymer type when measured under identicalsolvent and temperature conditions. Therefore, xanthan from X.campestris strain with multiple copies of gumB and gumC is of highermolecular weight compare to xanthan from control strain.

[0040] Methods: Five shake flasks each of the two broths were tested.The broths of each type were combined and the total volume measured. Thebroth was then precipitated in isopropyl alcohol. (Note: It wasestimated that the broth contained approximately 3% gum. Measuring thetotal broth volume and multiplying by 3% gave the approximate dry gumweight. This approximation was used to calculate the amount of waterrequired to produce approximately a 0.5% gum solution). The wet fibersof the precipitate were then immediately rehydrated with mixing in 0.01MNaCl to produce approximately a 0.5% gum solution. The fibers were mixedfor three hours with good shear using a 3-blade 2 inch diameterpropeller stirrer, then allowed to stand overnight. The followingprocedure was used to prepare the samples for intrinsic viscositymeasurements.

[0041] Filter the ˜0.5% gum solution, prepared above, using a GelmanScience 293 mm pressure filtration unit. The solution is first filteredthrough a 20 μ Magna nylon filter (N22SP29325) . The filter ispressurized to ˜60 psi, and the solution collected into clean beakers.(Note: the filters are changed when the flow rate is reduced to ˜5 dripsper minute.

[0042] Following the first filtration step, the samples are filtered twomore times using the above filtration unit. First, through a Millipore8.0 μ filter (SCWP 293 25), then through a Gelman Versapor® 293 mm 1.2 μfilter (66397). The filtered sample is recovered in clean beakersfollowing each filtration step.

[0043] After filtration, ˜600 ml of the gum solution is placed intoSpectra/Por® dialysis tubing 28.6 mm diameter Spectrum #S732706 (MWCO12,000 to 14,000). The tubing is cut into lengths of ˜18-20 inches, anda knot tied in one end. The solution is added to the tubing, filling itto within ˜2 inches from the end. Tie a second knot in the tubing suchthat as little air as possible is trapped in the tubing. Continue untilall the gum solution is in dialysis tubing.

[0044] Rinse the outside of the tubing containing the gum solution for˜1 minute with de-ionized water, then place the tubing into a containerof 0.1M NaCl. The salt solution should completely cover the dialysistubing.

[0045] Allow the tubing to sit in the 0.01M NaCl solution for 4 days,changing the NaCl solution daily. After the 4 days, cut open one end ofthe tubing and carefully transfer the gum solution to a clean beaker.

[0046] Solids are run on the filtered dialyzed solution using thefollowing procedure:

[0047] Using an analytical balance capable of weighing to ±0.0002 g,weigh and record the weight of a clean aluminum weighing dish VWR Cat#25433-008. (A)

[0048] Using a clean pipet add approximately 10 ml of the gum solutionto the aluminum pan and record the exact weight of the combined pan andgum solution. (B)

[0049] Place the pan with the solution into a 105° C. drying oven andallow to stand for 24 hours.

[0050] Remove the pan from the oven after 24 hours, cool and reweigh.Record the weight of the pan and remaining dried gum. (C)

[0051] Subtract the weight of the aluminum pan (A) from the weight ofthe pan plus the gum solution (B). Subtract the weight of the aluminumpan (A) from the weight of the dried gum plus the pan (C). Divide thefirst value (B-A) into the second (C-A). Multiply this value by 100 toobtain the % solids.

[0052] Note: Solids were run in triplicate for each filtered dialyzedsolution using the above procedure. The calculated % solids were thanaveraged for each sample and the averaged value was used.

[0053] Based on the solids determination for each solution, the samplesare diluted to 0.25% total gum concentration using 0.01M NaCl.

[0054] Intrinsic viscosity measurements were made using the VilasticViscoelasticity Analyzer (Vilastic Scientific, Inc., Austin, Tex.,fitted with the 0.0537 cm radius X 6.137 cm length tube. The instrumentwas calibrated with water prior to making measurements and verifiedafter the measurements were completed. Measurements were conducted usingthe instruments TIMET software protocol, set to a frequency of 2.0 Hz, aconstant strain of 1.0, and an integration time of 10 seconds. Thetemperature was maintained at 23.5° C. The samples were prepared bydilution of the 0.25% gum solution. Each dilution was mixed for 20minutes, and allowed to stand refrigerated overnight before beingmeasured. Six measurements were made for each dilution and averaged.Table 3 below shows the dilutions and the resultant averaged viscositiesfor each prepared sample. TABLE 3 Viscosity Dilutions Measurements 0.25%X.G. 0.01 M NaCl XWCM1 XWCM1/ Concentrations (ml) (ml) Control pBBR5-BCSolute 0.01 M 0 100 .921 .921 NaCl 0.0025% 1 99 1.114 1.165 0.0050% 2 981.326 1.486 0.0075% 3 97 1.537 1.829 0.0100% 4 96 1.762 2.181 0.0150% 694 2.302 2.963 0.0200% 8 92 2.920 3.901

[0055] Intrinsic viscosities were determined by plotting the reducedspecific viscosity (η_(sp)/c) against the gum concentrationη_(sp)/c=((η_(c)−η_(o))/η_(o)) where η_(c)=viscosit of the gum. Theintercept yields the intrinsic viscosit{tilde over (y)}. See FIG. 3.

[0056] The increase in intrinsic viscosity for the XWCM1/pBBR5-BCvariant is believed due to an increase in molecular weight. Intrinsicviscosity is proportional to the molecular weight for a given polymertype when measured under identical solvent and temperature conditions asdone in this experiment. The relationship between [η] and molecularweight is given by the Mark-Houwink equation [η] =kM^(a), where k and aare constants for a specified polymer type in a specified solvent at aspecified temperature. Because the constant “a” is positive number, anincrease in [η] can only be obtained by an increase in the molecularweight (M) unless the samples have a different molecular conformation inwhich case the Mark-Houwink equation is not obeyed.

Example 4 Procedure—Low Shear Rate Viscosity Measurement

[0057] Low shear rate viscosity measurements were performed on purifiedxanthan samples. The procedure used to measure LSRV is detailed below.Increased viscosity for xanthan from a strain with multiple copies ofgumB and gumC compared to xanthan from a control strain was observed.The data suggest that over-expression of both gumB and gumC is requiredfor increased chain length; over-expression of either gumB or gumCindividually is not sufficient to increase chain length.

[0058] Material and Equipment:

[0059] 1. Standard (synthetic) Tap Water (water containing 1000 ppm NaCland 40 ppm Ca⁺⁺ or 147 ppm CaCI₂0.2H2O): Prepare by dissolving in 20Liters of distilled water contained in a suitable container, 20 gm ofreagent grade NaCl and 2.94 gm of reagent grade CaCl₂0.2H₂O.

[0060] 2. Balance capable of accurately measuring to 0.01 gm.

[0061] 3. Brookfield LV Viscometer, Spindle #1, and spindle Guard.

[0062] 4. Standard laboratory glassware.

[0063] 5. Standard laboratory stirring bench. An RAE stirring motor(C25U) and stirring shaft ({fraction (5/16)}″) with 3-bladed propellermay be substituted.

[0064] Procedure:

[0065] 1. To 299 ml of synthetic tap water weighed in a 600 ml Berzelius(tall form) beaker, slowly add 0.75 gm (weighed to the nearest 0.01 gm)of product,

[0066] while stirring at 800 rpm.

[0067] 2. After stirring four hours at 800 rpm, remove the solution fromthe stirring bench, and allow to stand for 30 minutes.

[0068] 3. Adjust the temperature to room temperature and measure theviscosity using a Brookfield LV Viscometer with the No. 1 spindle at 3rpm. Record the viscosity after allowing the spindle to rotate for 3minutes.

Example 5 Quantification of Protein Expression

[0069] Cell lysates were subjected to Western blot and immunodetectionanalysis to establish the level of plasmid encoded GumB and GumC. Fourindependent blots were analyzed. Although absolute values for the samesample were not reproducible in each quantification, the relativequantities between samples remained the same in all the measurements.

[0070] Preparation of antibodies raised against GumB and GumC. An 1184bp DNA fragment encoding amino acid residues 53-447 of the GumC proteinwas produced by PCR amplification. The following primers were used:F2135: 5′GGAATTCCATATGTTGATGCCCGAGAAGTAC-3′ (SEQ ID NO: 4) and B3319:5′CGGGATCCTCAAAAGATCAGGCCCAACGCGAGG-3 (SEQ ID NO: 5)′. The PCR productwas digested with NdeI and BamHL subcloned into pET22b(+) and theresulting plasmid (pET-C) introduced into the E. coli strain BL21 (DE3).

[0071]E. coli BL21(pET-C) grown in L-broth containing 50 μgcarbenicillin ml⁻¹ to OD₆₀₀ 0.6 was induced with 1 mM IPTG for 3 h.Total cell lysates were prepared by treating with 1 mg lysozyme ml⁻¹ inlysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA pH8, 100 mM NaCl, 1 mM PMSF,0.1 mg DNase ml^(−1, 0.5)% Triton X-100) at 37° C. for 30 min, followedby sonication on ice. Cell debris was removed by low speedcentrifugation (Eppendof, 4000×g, 5 min) and the supernatant wasfractionated in a soluble and in a pellet (inclusion bodies) fraction bycentrifugation at 14000×g for 10 min. Pellet fraction was washed twicewith lysis buffer, in a volume identical to that of the original celllysate, once with 2 mg DOC ml⁻¹ in lysis buffer followed by three washeswith water. After treatment, proteins were separated by SDS-PAGE and themajor band containing the overproduced GumC protein was cut and elutedfor immunizing rabbits.

[0072]E. coli JM109(pQE-Xps#6, pREP4) grown in L-broth containing 50 μgcarbenicillin, 25 μg kanamycin ml⁻¹ to OD₆₀₀ 0.6 was induced with 1 mMIPTG for 3 h. Total cell lysates were prepared by treating with 1 mglysozyme ml⁻¹ in lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA pH8, 100 mMNaCl, 1 mM PMSF, 0.1 mg DNase ml⁻¹, 0.5% Triton X-100) at 37° C. for 30min, followed by sonication on ice. Cell debris was removed by low speedcentrifugation (Eppendof, 4000×g, 5 min) and the supernatant wasfractionated in a soluble and in an pellet (inclusion bodies) fractionby centrifugation at 14000×g for 10 min. Pellet fraction was washedtwice with lysis buffer, resuspended in 6 M guanidine hydrochloride in100 mM Phosphate buffer (pH7), 5 mM DTT, 5mM EDTA and inclusion bodieswere chromatographed on an FPLC Superdex HR200 (Pharmacia Biotech)pre-equilibrated with buffer D (4 M GdnHCl, 50 mM Phosphate buffer(pH7), 150 mM NaCl). Fractions containing GumB were pooled and used toimmunize mice.

[0073] Construction of plasmids pFD5, pBBR-promC, and pBBR5-B. A 3141 bpfragment containing gumB and gumC genes was obtained by partialdigestion of pIZD15-261 with BamHI (#318 and #3459) and cloned intoBamHI-digested pRK404 to yield plasmid pFD5. A 1480 bp fragment wasisolated by digestion of pGum02-19 with EcoRI (#1979) and BamHI (#3459)and cloned in pBBRlMCS-5 previously digested with the same enzymes toyield pBBR-promC. Digestion of pGum02-19 with HindIII in the MCS andEcoRI (#1979) produced a 1233 bp fragment, which was cloned inpBBR1MCS-5 to yield plasmid pBBR5-B.

[0074] New Zealand white female rabbits were immunized using GumCprepared as described above. A primary injection of 500 μg of theprotein with complete Freund's adjuvant was given to the rabbits,followed by three injections of 250 μg of the protein with incompleteadjuvant on alternate weeks. BALB/c female mice were immunized usingGumB prepared as described above. A primary injection of 100 μg of theprotein with complete Freund's adjuvant was given to the mice, followedby three injections of 50 μg of the protein with incomplete adjuvantonce a week. Polyclonal antibodies were prepared as described by Harlow& Lane ((1999) Using antibodies: a laboratory manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and antisera were stored at−70° C. To obtain GumC-specific antibodies, the serum was adsorbed withboth E. coli BL21(pET22b+) and Xc1231 acetone powders (Harlow & Lane,supra).

[0075] Protein extracts. Plasmids were introduced into the parentalstrain PRM-1 by electroporation. The resulting strains were grown in YMmedium at 28° C. and 250 rpm to middle-logarithmic phase. Cells wereharvested by centrifugation and the fresh-weight determined. The pelletwas washed twice with 10 mM Tris/HCl, 10 mM EDTA (pH 8.0) to removeexopolysaccharide and resuspended in the same buffer at a concentrationof 100 mg/ml. After addition of 100 μl Buffer A (10 mM Tris/HCl, 10 mMEDTA (pH 8.0), 1.5% SDS) to 50 μl of each sample, the mixture wasincubated at room temperature for 10 min followed by incubation at 100°C. for 12 min. Cell lysate was centrifuged at 14000×g (Eppendorf 5415 C)for 5 min and the supernatant collected was designated as total proteinextract. Protein concentration of each lysate was determined by themethod of Markwell ((1978) A modification of the Lowry procedure tosimplify protein determination in membrane and lipoprotein samples. AnalBiochem 87(1), 206-10) in the presence of SDS, using BSA as a standard.

[0076] SDS-PAGE and inmunodetection. Cell lysates (30 μg per lane) weremixed with sample buffer (125 mM Tris/HCl, pH6.8; 4% SDS, 20 mM DTT,0.05% bromophenol blue, 20% glycerol) and boiled for 2 min. Proteinswere separated by SDS-10% polyacrylamide gel according to the method ofSchagger and von Jagow ((1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Analytical Biochemistry 166(2),368-79). Electroblotting was performed using a semi-dry transfer system(Hoefer Semiphor unit) onto Immobilon-P membranes (PVDF, Millipore). Thetransfer was performed in a buffer containing 10 mM CAPS (pH11), 10%(v/v) methanol for 30 min at 2.5 mA/cm² of gel surface area. Once theelectrotransfer was complete, the blots were stained with 0.5% Ponceau-Sred to assess the quality of the transfer and washed with Milli-Q®-gradewater. The blots were blocked overnight at 4° C. with 5% nonfat milkpowder in TBST (150 mM NaCl, 10 mM Tris/HCl pH8, 0.05% Tween-20) (Harlow& Lane, supra) and then incubated with anti-GumB (1:3000) or anti-GumC(1:5000) antibodies in 3% nonfat milk powder in TBST at room temperaturefor 3 h. Alkaline phosphatase-conjugated goat anti-mouse IgG oranti-rabbit IgG (Sigma) were used for detection, respectively, asdescribed by the manufacturer. The blots were washed three times withTBST and were developed in a solution containing nitrobluetetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP, Promega).Commercial protein markers MW-SDS-70L (Sigma) were used to calibrateSDS-PAGE.

[0077] Blot quantification. The intensities of GumB and GumC proteinbands were determined by scanning the NBT/BCIP developed filters with aUVP Densitometer (Ultra Violet Products) and quantified with GelWorks IDAnalysis software (NonLinear Dynamics Ltd). Each filter contained areference lane of a PRM-1(pBBR-prom) extract to establish the level ofchromosomally encoded GumB and GumC in the wild type cells. Relativeamounts of GumB and GumC were observed. See FIGS. 2A and 2B.

Example 6 Procedure—Molecular Length or Weight Determination UsingAtomic Force Microscopy

[0078] The direct visualization technique called Atomic force Microscopy(AFM) or Scanning Probe Microscope (SPM) was used to image the lengthsof xanthan molecules from X. campestris strains with (XWCM1/pBBR5-BC)and without (XWCM1) multiple copies of gumB and gumC. The procedure usedto perform AFM is detailed below. We observed that the average molecularcontour length of xanthan molecules produced by a strain with multiplecopies of gumB and gumC was much longer than that of the parentalstrain.

[0079] A 0.1 wt % of gum solution was prepared by mixing 0.1 g of gum in100 gram distilled water for ˜3 hours. A 1-ppm stock solution wasprepared by diluting 20 μl of the 0.1 wt % solution into a 20 g 0.1Mammonium acetate solution. 20 μl of the 1 ppm stock solution was sprayedonto freshly cleaved mica disc(s) (˜1 cm²). These mica sample disc(s)were then placed in a heated (˜60° C.) vacuum chamber for ˜one hour toremove excess water. The dried mica disc(s) were then scanned using theTapping Mode of the AFM. The molecular contour length of all AFM imageswas measured with the software provided by Digital Instruments.

[0080] Contour lengths of population of xanthan molecules were measured.The results of this study are summarized in Table 4. (Molecules in eachsize class are less than or equal to the length indicated; the number ofmolecules indicated in a size class do not include the molecules countedin a smaller size class.) These results demonstrated that xanthanmolecules from X. campestris strain with multiple copies of gumB andgumC were significantly larger then xanthan molecules from controlstrain. The atomic force microscopy (AFM) or scanning probe microscopy(SPM) was performed with a commercial instrument (Nanoscope IIIa,Digital Instruments, Santa Barbara, Calif.) using a silicon nitridecantilever tip. TABLE 4 AFM Measurement of Xanthan Molecules ContourLength XWCM1 XWCM1/pBBR5-BC Length* Molecules Frequency DistributionLength Molecules Frequency Distribution (μm) (count) (%) No. Avg. Wt.Avg. (μm) (count) (%) No. Avg. Wt. Avg. 0.5 225 51.5 ≦3 μm = ≦3 μm = 0.5150 28.4 ≦3 μm = ≦3 μm = 1 130 29.7 99.8% 98.7% 1 163 30.9 90.9% 70.9%1.5 40 9.2 1.5 82 15.5 2 25 5.7 2 44 8.3 2.5 13 3.0 2.5 29 5.5 3 3 0.7 312 2.3 3.5 0 0.0 >3 μm = >3 μm = 3.5 12 2.3 >3 μm = >3 μm = 4 0 0.0 0.2%1.3% 4 13 2.5 9.1% 29.1% 4.5 0 0.0 4.5 7 1.3 5 1 0.2 5 4 0.8 5.5 0 0.05.5 4 0.8 6 0 0.0 6 0 0.0 6.5 0 0.0 6.5 3 0.6 7 0 0.0 7 2 0.4 7.5 0 0.07.5 0 0.0 8 0 0.0 8 0 0.0 8.5 0 0.0 8.5 1 0.2 9 0 0.0 9 1 0.2 9.5 0 0.09.5 1 0.2 10 0 0.0 10 0 0.0 Total 437 Total 528

Example 7 Evaluation of Seawater Viscosity

[0081] Xanthan produced by strain XWCM-1/pBBR5-BC was evaluated forseawater vi

[0082] scosity (SWV), compared to a commercial xanthan product(Xanvis™). Typical SWV for Xanvis™ xanthan product is in the range of 18to 22.

[0083] Seawater viscosity was determined using the following procedure.Seawater solution was prepared by dissolving 41.95 g of sea salt (ASTMD1141-52, from Lake Products Co., Inc. Maryland Heights, Mo.) in 1 literdeionized water. 300 ml of seawater solution was transferred to a mixingcup that was attached to a Hamilton-Beach 936-2 mixer (Hamilton-BeachDiv., Washington, D.C.). The mixer speed control was set to low and asingle fluted disk attached to the mixing shaft. At the low speedsetting, the mixer shaft rotates at approximately 4,000-6,000 rpm. 0.86g of biogum product was slowly added over 15-30 seconds to the mixingcup and allowed to mix for 5 minutes. The mixer speed control was set tohigh (11,000±1,000 rpm) and the test solution was allowed to mix forapproximately 5 minutes. The mixture was allowed to mix for a total of45 minutes, starting from time of biogum product addition. At the end ofthe 45 minutes mixing time, 2-3 drops of Bara Defoam (NL Baroid/NLindustries, Inc., Houston, Tex.) was added and stirring was continuedfor an additional 30 seconds.

[0084] The mixing cup was removed from the mixer and immersed in chilledwater to lower the fluid's temperature to 25±0.5° C. In order to insurea homogeneous solution, the solution was re-mixed after cooling for 5seconds at 11,000±1,000 rpm. The solution was transferred from themixing cup to 400 ml Pyrex beaker and Fann viscosity (Fann Viscometer,Model 35A) was measured. This was accomplished by mixing at low speed(about 3 rpm). The reading was allowed to stabilize and then the shearstress value was read from dial and recorded as the SWV value at 3 rpm.TABLE 5 Quality of XWCM-1/pBBR5-BC xanthan and Xanvis ™ xanthan SWVSample DR^(a) XWCM-1/pBBR5-BC 29 30 Xanvis ™ xanthan 22

[0085] References

[0086] 1. Kidby, D., Sandford, P., Herman, A., and Cadmus, M. (1977)Maintenance procedures for the curtailment of genetic instability:Xanthomonas campestris NRRL B-1459. Applied and EnvironmentalMicrobiology 33(4), 840-5

[0087] 2. Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X.W., Finlay, D. R., Guiney, D., and Helinski, D. R. (1985) Plasmidsrelated to the broad host range vector, pRK290, useful for gene cloningand for monitoring gene expression. Plasmid 13(2), 149-53

[0088] 3. Simon, R., Priefer U. and Puhler A. (1983) A broad host rangemobilization system for in vivo genetic engineering: transposonmutagenesis in Gram-negative bacteria. Biotechnology 1, 784-791

[0089] 4. Harding, N. E., Cleary, J. M., Cabanas, D. K., Rosen, I. G.,and Kang, K. S. (1987) Genetic and physical analyses of a cluster ofgenes essential for xanthan gumBiosynthesis in Xanthomonas campestris. JBacteriol 169(6), 2854-61.

[0090] 5. Katzen, F., Becker, A., Zorreguieta, A., Puhler, A., andIelpi, L. (1996) Promoter analysis of the Xanthomonas campestris pv.campestris gum operon directing biosynthesis of the xanthanpolysaccharide. J Bacteriol 178(14), 4313-8.

[0091] 6. Capage, M. R., D. H. Doherty, M. R. Betlach, and R. W.Vanderslice. (1987) Recombinant-DNA mediated production of xanthan gum.International patent WO87/05938.

[0092] 7. Becker, A., Niehaus, K., and Puhler, A. (1995)Low-molecular-weight succinoglycan is predominantly produced byRhizobium meliloti strains carrying a mutated ExoP protein characterizedby a periplasmic N-terminal domain and a missing C-terminal domain.Molecular Microbiology 16(2), 191-203

[0093] 8. Schafer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach,G., and Puhler, A. (1994) Small mobilizable multi-purpose cloningvectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacteriumglutarnicum. Gene 145(1), 69-73

[0094] 9. Yanisch_Perron, C., Vieira, J., and Messing, J. (1985)Improved M13 phage cloning vectors and host strains: nucleotidesequences of the M13mp18 and pUC19 vectors. Gene 33(1), 103-19

[0095] 10. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T.,Farris, M. A., Roop, R. M., and Peterson, K. M. (1995) Four newderivatives of the broad-host-range cloning vector pBBR1MCS, carryingdifferent antibiotic-resistance cassettes. Gene 166(1), 175-6

[0096] 11. Harding, N. E., Cleary, J. M., Cabanas, D. K., Rosen, I. G.,and Kang, K. S. (1987) Genetic and physical analyses of a cluster ofgenes essential for xanthan gumBiosynthesis in Xanthomonas campestris.Journal of Bacteriology 169(6), 2854-61

[0097] 12. Harding N. E., R. S., Raimondi A., Cleary J. M and Ielpi L.(1993) Identification, genetic and biochemical analysis of genesinvolved in synthesis sugar nucleotide precursors of xanthan gum. J.Gen. Microbiol 139, 447-457

[0098] 13. Sambrook, J., and Russell, D. W. (2001) Molecular cloning: alaboratory manual, 3rd Ed., Cold Spring Harbor, N.Y. Cold Spring HarborLaboratory Press, 2001.

1 5 1 7604 DNA Xanthomomas campestris CDS (2715)...(4133) gumC 1accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg 120aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180aaaagtgcca cctggcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg 240atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact 300tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct 360tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc 420gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc 480tcactacgcg gctgctcaaa cctgggcaga acgtaagccg cgagagcgcc aacaaccgct 540tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa 600tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga 660tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg 720cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg 780ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc 840ttgctgcttg gatgcccgag gcatagactg tacaaaaaaa cagtcataac aagccatgaa 900aaccgccact gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga 960gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg 1020tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat attatacgca aggcgacaag 1080gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 1140agaatgctta atgaattaca acagttttta tgcatgcgcc caatacgcaa accgcctctc 1200cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 1260ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 1320cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 1380ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa 1440aagctgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttgc atgcctgcag 1500gtcgactcta gtggtcgtcg gttcgaatcc ggctaccccg accaaacaac aggcctacgt 1560cgcaagacgt gggccttttt gttgcgtcgc aacatgtcag ttcgatggca ttccaggcta 1620tgccactatg cgcaacggca tattgcaagg cggcatatgc aagtcctgta cgcaattatt 1680tcgcggttca ggctgctaca agtcgggatc agcaggcgtc cgtaagtgcc cggaaacgct 1740agagttcgta tgctgagaat gacgacccag gtcacgttct cttaacgtcg aggcgacgaa 1800cttgaatcaa taggccaacg ccgtcaaaaa aatggcgtgt tgtgccttgc gatgtgttcg 1860ttctatgcca tagtgcactg caacacgcga ttcaacgttg gtcccggcac gcgtcgggat 1920gcaacttcct gtcgtacgtt cgtgctggcg cctgagccgg ttgaatgctg cgcgaggtcc 1980tgtcccaccc aacagaggca gccagctaca cgc atg aag aaa ctg atc gga cga 2034Met Lys Lys Leu Ile Gly Arg 1 5 ctc tgc caa ggc ctc agc ctg gct ctg ctctgc tcg atg tcg ctg ggc 2082 Leu Cys Gln Gly Leu Ser Leu Ala Leu Leu CysSer Met Ser Leu Gly 10 15 20 gct tgc agc acc ggc ccg gag atg gcg tct tcgctg ccg cat ccg gac 2130 Ala Cys Ser Thr Gly Pro Glu Met Ala Ser Ser LeuPro His Pro Asp 25 30 35 ccg ctg gca atg tcc acg gtg cag ccc gaa tac cgtctt gcg ccg ggc 2178 Pro Leu Ala Met Ser Thr Val Gln Pro Glu Tyr Arg LeuAla Pro Gly 40 45 50 55 gat ctg ttg ctg gtg aag gtg ttt cag atc gac gatctg gag cgg cag 2226 Asp Leu Leu Leu Val Lys Val Phe Gln Ile Asp Asp LeuGlu Arg Gln 60 65 70 gtc cgc atc gac cag aac ggt cac atc tca ctg ccg ttgatt ggc gac 2274 Val Arg Ile Asp Gln Asn Gly His Ile Ser Leu Pro Leu IleGly Asp 75 80 85 gtc aag gcc gcc ggt ctg ggc gtt ggc gaa ctg gaa aag ctggtc gcc 2322 Val Lys Ala Ala Gly Leu Gly Val Gly Glu Leu Glu Lys Leu ValAla 90 95 100 gat cgg tat cgc gca ggc tac ctg cag cag ccg cag att tcggta ttc 2370 Asp Arg Tyr Arg Ala Gly Tyr Leu Gln Gln Pro Gln Ile Ser ValPhe 105 110 115 gtg cag gag tcc aac ggg cgt cgc gtc acg gtc act ggt gcggta gac 2418 Val Gln Glu Ser Asn Gly Arg Arg Val Thr Val Thr Gly Ala ValAsp 120 125 130 135 gag ccg ggc atc tac ccg gtg atc ggc gcc aac ctc accttg cag cag 2466 Glu Pro Gly Ile Tyr Pro Val Ile Gly Ala Asn Leu Thr LeuGln Gln 140 145 150 gcg atc gcg cag gcc aag ggt gtc agc acg gtg gca agccgc ggc aac 2514 Ala Ile Ala Gln Ala Lys Gly Val Ser Thr Val Ala Ser ArgGly Asn 155 160 165 gtg atc gtg ttc cgc atg gtc aac ggg caa aaa atg attgcg cgg ttc 2562 Val Ile Val Phe Arg Met Val Asn Gly Gln Lys Met Ile AlaArg Phe 170 175 180 gac ctg acc gag atc gag aag ggg gcc aat ccg gat cctgag att tat 2610 Asp Leu Thr Glu Ile Glu Lys Gly Ala Asn Pro Asp Pro GluIle Tyr 185 190 195 ggc ggc gac att gtc gtg gtg tat cgc tcg gat gcg cgcgtg tgg ttg 2658 Gly Gly Asp Ile Val Val Val Tyr Arg Ser Asp Ala Arg ValTrp Leu 200 205 210 215 cgc acc atg ctg gaa ctg acc ccc ttg gtg atg gtgtgg cgc gct tac 2706 Arg Thr Met Leu Glu Leu Thr Pro Leu Val Met Val TrpArg Ala Tyr 220 225 230 cga tga gt atg aat tca gac aat cgt tcc tct tcgtcg cag cgt cat 2753 Arg * Met Asn Ser Asp Asn Arg Ser Ser Ser Ser GlnArg His 235 240 245 ggt cat ctg gaa ctg gca gat gtc gac ttg atg gac tactgg cgc gcc 2801 Gly His Leu Glu Leu Ala Asp Val Asp Leu Met Asp Tyr TrpArg Ala 250 255 260 ctg gtc tcg cag ctc tgg ctg atc atc ctg atc gcc gtcggc gcg ctg 2849 Leu Val Ser Gln Leu Trp Leu Ile Ile Leu Ile Ala Val GlyAla Leu 265 270 275 ttg ctg gca ttc ggc atc acg atg ttg atg ccc gag aagtac cgc gcc 2897 Leu Leu Ala Phe Gly Ile Thr Met Leu Met Pro Glu Lys TyrArg Ala 280 285 290 acc agc acc ctg cag atc gaa cgt gac tcg ctc aat gtggtg aac gtc 2945 Thr Ser Thr Leu Gln Ile Glu Arg Asp Ser Leu Asn Val ValAsn Val 295 300 305 gac aac ctg atg ccg gtg gaa tcg ccg cag gat cgc gatttc tac cag 2993 Asp Asn Leu Met Pro Val Glu Ser Pro Gln Asp Arg Asp PheTyr Gln 310 315 320 325 acc cag tac cag ttg ctg cag agc cgt tcg ctg gcgcgt gcg gtg atc 3041 Thr Gln Tyr Gln Leu Leu Gln Ser Arg Ser Leu Ala ArgAla Val Ile 330 335 340 cgg gaa gcc aag ctc gat cag gag ccg gcg ttc aaggag cag gtg gag 3089 Arg Glu Ala Lys Leu Asp Gln Glu Pro Ala Phe Lys GluGln Val Glu 345 350 355 gag gcg ctg gcc aaa gcc gcc gaa aag aat ccc gaggcg ggt aag tcg 3137 Glu Ala Leu Ala Lys Ala Ala Glu Lys Asn Pro Glu AlaGly Lys Ser 360 365 370 ctc gat tcg cgg cag gcg atc gtc gag cgc agc ctcacc gat acg ttg 3185 Leu Asp Ser Arg Gln Ala Ile Val Glu Arg Ser Leu ThrAsp Thr Leu 375 380 385 ctc gcc ggg ctg gtg gtc gag ccg atc ctc aac tcgcgc ctg gtg tac 3233 Leu Ala Gly Leu Val Val Glu Pro Ile Leu Asn Ser ArgLeu Val Tyr 390 395 400 405 gtc aat tac gat tcg cca gac ccg gtg ctg gccgcc aag atc gcc aat 3281 Val Asn Tyr Asp Ser Pro Asp Pro Val Leu Ala AlaLys Ile Ala Asn 410 415 420 acg tac ccg aag gtg ttc atc gtc agc acc caggaa cgc cgc atg aag 3329 Thr Tyr Pro Lys Val Phe Ile Val Ser Thr Gln GluArg Arg Met Lys 425 430 435 gcg tct tcg ttt gcg aca cag ttt ctg gct gagcgc ctg aag cag ttg 3377 Ala Ser Ser Phe Ala Thr Gln Phe Leu Ala Glu ArgLeu Lys Gln Leu 440 445 450 cgc gag aag gtc gaa gac tct gaa aag gat ctggtc tcg tat tcg acc 3425 Arg Glu Lys Val Glu Asp Ser Glu Lys Asp Leu ValSer Tyr Ser Thr 455 460 465 gaa gag cag atc gtg tcg gtt ggc gat gac aagccc tcg ctg cct gcg 3473 Glu Glu Gln Ile Val Ser Val Gly Asp Asp Lys ProSer Leu Pro Ala 470 475 480 485 cag aat ctg acc gat ctc aat gcg ttg ctggca tcc gca cag gac gcc 3521 Gln Asn Leu Thr Asp Leu Asn Ala Leu Leu AlaSer Ala Gln Asp Ala 490 495 500 cgg atc aag gcc gag tca gct tgg cgg caggct tcc agt ggc gat ggc 3569 Arg Ile Lys Ala Glu Ser Ala Trp Arg Gln AlaSer Ser Gly Asp Gly 505 510 515 atg tca ttg ccg cag gtg ttg agc agc ccgctg att caa agc ctg cgc 3617 Met Ser Leu Pro Gln Val Leu Ser Ser Pro LeuIle Gln Ser Leu Arg 520 525 530 agc gag cag gtg cgt ctg acc agc gag taccag cag aaa ctg tcg acc 3665 Ser Glu Gln Val Arg Leu Thr Ser Glu Tyr GlnGln Lys Leu Ser Thr 535 540 545 ttc aag ccg gat tac ccg gag atg cag cgcctc aag gcg cag atc gaa 3713 Phe Lys Pro Asp Tyr Pro Glu Met Gln Arg LeuLys Ala Gln Ile Glu 550 555 560 565 gag tcg cgt cgt cag atc aat ggc gaagtc atc aat atc cgt cag tcg 3761 Glu Ser Arg Arg Gln Ile Asn Gly Glu ValIle Asn Ile Arg Gln Ser 570 575 580 ctg aag gcg acc tac gac gcc tcc gtgcat cag gag cag ctg ctc aac 3809 Leu Lys Ala Thr Tyr Asp Ala Ser Val HisGln Glu Gln Leu Leu Asn 585 590 595 gac cgc atc gcc ggt ctg cgg tcc aacgag ctg gat ctg cag agc cgc 3857 Asp Arg Ile Ala Gly Leu Arg Ser Asn GluLeu Asp Leu Gln Ser Arg 600 605 610 agc atc cgc tac aac atg ctc aag cgcgac gtc gac acc aac cgc cag 3905 Ser Ile Arg Tyr Asn Met Leu Lys Arg AspVal Asp Thr Asn Arg Gln 615 620 625 ctc tac gat gcg ctc ctg cag cgc tacaag gaa atc ggc gtg gcg agc 3953 Leu Tyr Asp Ala Leu Leu Gln Arg Tyr LysGlu Ile Gly Val Ala Ser 630 635 640 645 aac gtg ggc gcc aac aac gtg accatc gtc gat acc gca gac gtg cct 4001 Asn Val Gly Ala Asn Asn Val Thr IleVal Asp Thr Ala Asp Val Pro 650 655 660 acg tct aag act tcg ccg aaa ctcaaa ttg aac ctc gcg ttg ggc ctg 4049 Thr Ser Lys Thr Ser Pro Lys Leu LysLeu Asn Leu Ala Leu Gly Leu 665 670 675 atc ttt ggc gta ttc ctg ggc gtggct gtg gct ctg gtt cgc tac ttc 4097 Ile Phe Gly Val Phe Leu Gly Val AlaVal Ala Leu Val Arg Tyr Phe 680 685 690 ctg cgt ggg cct tct ccg agg tcgcgg ttg aac tga catcgtgatg 4143 Leu Arg Gly Pro Ser Pro Arg Ser Arg LeuAsn * 695 700 ttgcaaaacg atggttaatt gaagtgacaa ctgattcagc gtggaaaaggtgggatcccg 4203 taaggtgcgg gctccctcgt ttgaaggttt gtctctgttg aaacaaagggctgtcgtgcg 4263 atctggggtc ggtaggtatt accgcggtga tcggacgaca ggatgattgaaagctcgcgt 4323 gcgattcgta tgttcccccg catgcctgca ggtcgactct agagcggccgccaccgcggt 4383 ggagctccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggccgtcgttttac 4443 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgcagcacatcccc 4503 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcccaacagttgc 4563 gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaattcgcgttaaa 4623 tttttgttaa atcagctcat tttttaacca ataggccgac tgcgatgagtggcagggcgg 4683 ggcgtaattt ttttaaggca gttattggtg cccttaaacg cctggtgctacgcctgaata 4743 agtgataata agcggatgaa tggcagaaat tcgaaagcaa attcgacccggtcgtcggtt 4803 cagggcaggg tcgttaaata gccgcttatg tctattgctg gtttaccggtttattgacta 4863 ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgctcaggctctcc 4923 ccgtggaggt aataattgac gatatgatca tttattctgc ctcccagagcctgataaaaa 4983 cggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtggcccggctcca 5043 tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgaggcggctacagcc 5103 gatagtctgg aacagcgcac ttacgggttg ctgcgcaacc caagtgctaccggcgcggca 5163 gcgtgacccg tgtcggcggc tccaacggct cgccatcgtc cagaaaacacggctcatcgg 5223 gcatcggcag gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtcaaggctggca 5283 ggtctggttc catgcccgga atgccgggct ggctgggcgg ctcctcgccggggccggtcg 5343 gtagttgctg ctcgcccgga tacagggtcg ggatgcggcg caggtcgccatgccccaaca 5403 gcgattcgtc ctggtcgtcg tgatcaacca ccacggcggc actgaacaccgacaggcgca 5463 actggtcgcg gggctggccc cacgccacgc ggtcattgac cacgtaggccgacacggtgc 5523 cggggccgtt gagcttcacg acggagatcc agcgctcggc caccaagtccttgactgcgt 5583 attggaccgt ccgcaaagaa cgtccgatga gcttggaaag tgtcttctggctgaccacca 5643 cggcgttctg gtggcccatc tgcgccacga ggtgatgcag cagcattgccgccgtgggtt 5703 tcctcgcaat aagcccggcc cacgcctcat gcgctttgcg ttccgtttgcacccagtgac 5763 cgggcttgtt cttggcttga atgccgattt ctctggactg cgtggccatgcttatctcca 5823 tgcggtaggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaacttttcggcagc 5883 gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttctttaacctac 5943 gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtaccccccttttttaagtt 6003 gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgcccaaagaagg 6063 gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggcaaaaagtggcc 6123 cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtggcgctgcccc 6183 cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgggcttcgcctt 6243 cgactgcccc cactcgcata ggcttgggtc gttccaggcg cgtcaaggccaagccgctgc 6303 gcggtcgctg cgcgagcctt gacccgcctt ccacttggtg tccaaccggcaagcgaagcg 6363 cgcaggccgc aggccggagg cttttcccca gagaaaatta aaaaaattgatggggcaagg 6423 ccgcaggccg cgcagttgga gccggtgggt atgtggtcga aggctgggtagccggtgggc 6483 aatccctgtg gtcaagctcg tgggcaggcg cagcctgtcc atcagcttgtccagcagggt 6543 tgtccacggg ccgagcgaag cgagccagcc ggtggccgct cgcggccatcgtccacatat 6603 ccacgggctg gcaagggagc gcagcgaccg cgcagggcga agcccggagagcaagcccgt 6663 agggcgccgc agccgccgta ggcggtcacg actttgcgaa gcaaagtctagtgagtatac 6723 tcaagcattg agtggcccgc cggaggcacc gccttgcgct gcccccgtcgagccggttgg 6783 acaccaaaag ggaggggcag gcatggcggc atacgcgatc atgcgatgcaagaagctggc 6843 gaaaatgggc aacgtggcgg ccagtctcaa gcacgcctac cgcgagcgcgagacgcccaa 6903 cgctgacgcc agcaggacgc cagagaacga gcactgggcg gccagcagcaccgatgaagc 6963 gatgggccga ctgcgcgagt tgctgccaga gaagcggcgc aaggacgctgtgttggcggt 7023 cgagtacgtc atgacggcca gcccggaatg gtggaagtcg gccagccaagaacagcaggc 7083 ggcgttcttc gagaaggcgc acaagtggct ggcggacaag tacggggcggatcgcatcgt 7143 gacggccagc atccaccgtg acgaaaccag cccgcacatg accgcgttcgtggtgccgct 7203 gacgcaggac ggcaggctgt cggccaagga gttcatcggc aacaaagcgcagatgacccg 7263 cgaccagacc acgtttgcgg ccgctgtggc cgatctaggg ctgcaacggggcatcgaggg 7323 cagcaaggca cgtcacacgc gcattcaggc gttctacgag gccctggagcggccaccagt 7383 gggccacgtc accatcagcc cgcaagcggt cgagccacgc gcctatgcaccgcagggatt 7443 ggccgaaaag ctgggaatct caaagcgcgt tgagacgccg gaagccgtggccgaccggct 7503 gacaaaagcg gttcggcagg ggtatgagcc tgccctacag gccgccgcaggagcgcgtga 7563 gatgcgcaag aaggccgatc aagcccaaga gacggcccga g 7604 2 232PRT Xanthomomas campestris 2 Met Lys Lys Leu Ile Gly Arg Leu Cys Gln GlyLeu Ser Leu Ala Leu 1 5 10 15 Leu Cys Ser Met Ser Leu Gly Ala Cys SerThr Gly Pro Glu Met Ala 20 25 30 Ser Ser Leu Pro His Pro Asp Pro Leu AlaMet Ser Thr Val Gln Pro 35 40 45 Glu Tyr Arg Leu Ala Pro Gly Asp Leu LeuLeu Val Lys Val Phe Gln 50 55 60 Ile Asp Asp Leu Glu Arg Gln Val Arg IleAsp Gln Asn Gly His Ile 65 70 75 80 Ser Leu Pro Leu Ile Gly Asp Val LysAla Ala Gly Leu Gly Val Gly 85 90 95 Glu Leu Glu Lys Leu Val Ala Asp ArgTyr Arg Ala Gly Tyr Leu Gln 100 105 110 Gln Pro Gln Ile Ser Val Phe ValGln Glu Ser Asn Gly Arg Arg Val 115 120 125 Thr Val Thr Gly Ala Val AspGlu Pro Gly Ile Tyr Pro Val Ile Gly 130 135 140 Ala Asn Leu Thr Leu GlnGln Ala Ile Ala Gln Ala Lys Gly Val Ser 145 150 155 160 Thr Val Ala SerArg Gly Asn Val Ile Val Phe Arg Met Val Asn Gly 165 170 175 Gln Lys MetIle Ala Arg Phe Asp Leu Thr Glu Ile Glu Lys Gly Ala 180 185 190 Asn ProAsp Pro Glu Ile Tyr Gly Gly Asp Ile Val Val Val Tyr Arg 195 200 205 SerAsp Ala Arg Val Trp Leu Arg Thr Met Leu Glu Leu Thr Pro Leu 210 215 220Val Met Val Trp Arg Ala Tyr Arg 225 230 3 472 PRT Xanthomomas campestris3 Met Asn Ser Asp Asn Arg Ser Ser Ser Ser Gln Arg His Gly His Leu 1 5 1015 Glu Leu Ala Asp Val Asp Leu Met Asp Tyr Trp Arg Ala Leu Val Ser 20 2530 Gln Leu Trp Leu Ile Ile Leu Ile Ala Val Gly Ala Leu Leu Leu Ala 35 4045 Phe Gly Ile Thr Met Leu Met Pro Glu Lys Tyr Arg Ala Thr Ser Thr 50 5560 Leu Gln Ile Glu Arg Asp Ser Leu Asn Val Val Asn Val Asp Asn Leu 65 7075 80 Met Pro Val Glu Ser Pro Gln Asp Arg Asp Phe Tyr Gln Thr Gln Tyr 8590 95 Gln Leu Leu Gln Ser Arg Ser Leu Ala Arg Ala Val Ile Arg Glu Ala100 105 110 Lys Leu Asp Gln Glu Pro Ala Phe Lys Glu Gln Val Glu Glu AlaLeu 115 120 125 Ala Lys Ala Ala Glu Lys Asn Pro Glu Ala Gly Lys Ser LeuAsp Ser 130 135 140 Arg Gln Ala Ile Val Glu Arg Ser Leu Thr Asp Thr LeuLeu Ala Gly 145 150 155 160 Leu Val Val Glu Pro Ile Leu Asn Ser Arg LeuVal Tyr Val Asn Tyr 165 170 175 Asp Ser Pro Asp Pro Val Leu Ala Ala LysIle Ala Asn Thr Tyr Pro 180 185 190 Lys Val Phe Ile Val Ser Thr Gln GluArg Arg Met Lys Ala Ser Ser 195 200 205 Phe Ala Thr Gln Phe Leu Ala GluArg Leu Lys Gln Leu Arg Glu Lys 210 215 220 Val Glu Asp Ser Glu Lys AspLeu Val Ser Tyr Ser Thr Glu Glu Gln 225 230 235 240 Ile Val Ser Val GlyAsp Asp Lys Pro Ser Leu Pro Ala Gln Asn Leu 245 250 255 Thr Asp Leu AsnAla Leu Leu Ala Ser Ala Gln Asp Ala Arg Ile Lys 260 265 270 Ala Glu SerAla Trp Arg Gln Ala Ser Ser Gly Asp Gly Met Ser Leu 275 280 285 Pro GlnVal Leu Ser Ser Pro Leu Ile Gln Ser Leu Arg Ser Glu Gln 290 295 300 ValArg Leu Thr Ser Glu Tyr Gln Gln Lys Leu Ser Thr Phe Lys Pro 305 310 315320 Asp Tyr Pro Glu Met Gln Arg Leu Lys Ala Gln Ile Glu Glu Ser Arg 325330 335 Arg Gln Ile Asn Gly Glu Val Ile Asn Ile Arg Gln Ser Leu Lys Ala340 345 350 Thr Tyr Asp Ala Ser Val His Gln Glu Gln Leu Leu Asn Asp ArgIle 355 360 365 Ala Gly Leu Arg Ser Asn Glu Leu Asp Leu Gln Ser Arg SerIle Arg 370 375 380 Tyr Asn Met Leu Lys Arg Asp Val Asp Thr Asn Arg GlnLeu Tyr Asp 385 390 395 400 Ala Leu Leu Gln Arg Tyr Lys Glu Ile Gly ValAla Ser Asn Val Gly 405 410 415 Ala Asn Asn Val Thr Ile Val Asp Thr AlaAsp Val Pro Thr Ser Lys 420 425 430 Thr Ser Pro Lys Leu Lys Leu Asn LeuAla Leu Gly Leu Ile Phe Gly 435 440 445 Val Phe Leu Gly Val Ala Val AlaLeu Val Arg Tyr Phe Leu Arg Gly 450 455 460 Pro Ser Pro Arg Ser Arg LeuAsn 465 470 4 31 DNA Xanthomomas campestris 4 ggaattccat atgttgatgcccgagaagta c 31 5 33 DNA Xanthomomas campestris 5 cgggatcctc aaaagatcaggcccaacgcg agg 33

1. An unpasteurized xanthan composition from a cell which over-expressesgumB and gumC, wherein said composition has an intrinsic viscosity whichis at least 20% greater than xanthan from a corresponding strain whichdoes not over-express gumB and gumC.
 2. The unpasteurized xanthancomposition of claim 1 which has an intrinsic viscosity which is atleast 25% greater than xanthan from the corresponding strain.
 3. Theunpasteurized xanthan composition of claim 1 which has an intrinsicviscosity which is at least 30% greater than xanthan from thecorresponding strain.
 4. A xanthan composition comprising a populationof xanthan molecules having a range of molecular lengths, wherein atleast 1% of the population has a length of at least 3 um as measured byatomic force microscopy.
 5. The method of claim 4 wherein at least 5% ofthe population has a length of at least 3 um as measured by atomic forcemicroscopy.
 6. A xanthan composition comprising a population of xanthanmolecules having a range of molecular lengths, wherein at least 1% ofthe population has a length of at least 4 um as measured by atomic forcemicroscopy.
 7. The xanthan composition of claim 6 wherein at least 1% ofthe population has a length of at least 5 um.
 8. The xanthan compositionof claim 6 wherein at least 1% of the population has a length of atleast 7 um.
 9. A xanthan composition comprising a population of xanthanmolecules having a range of molecular lengths, wherein at least 5% ofthe total mass of the xanthan molecules in the composition is due toxanthan molecules having a molecular length greater than 3 um asmeasured by atomic force microscopy.
 10. The xanthan composition ofclaim 9 wherein at least 10% of the total mass of the xanthan moleculesin the composition is due to xanthan molecules having a molecular lengthgreater than 3 um as measured by atomic force microscopy.
 11. Thexanthan composition of claim 9 wherein at least 15% of the total mass ofthe xanthan molecules in the composition is due to xanthan moleculeshaving a molecular length greater than 3 um as measured by atomic forcemicroscopy.
 12. The xanthan composition of claim 9 wherein at least 20%of the total mass of the xanthan molecules in the composition is due toxanthan molecules having a molecular length greater than 3 um asmeasured by atomic force microscopy.
 13. A food product comprising axanthan composition according to claim
 1. 14. A food product comprisinga xanthan composition according to claim
 4. 15. A food productcomprising a xanthan composition according to claim
 6. 16. A foodproduct comprising a xanthan composition according to claim
 9. 17. Thefood product of claim 1, claim 4, claim 6, or claim 9 wherein the foodis selected from the group consisting of a salad dressing, a syrup, ajuice drink, and a frozen dessert.
 18. A printing dye comprising axanthan composition according to claim
 1. 19. A printing dye comprisinga xanthan composition according to claim
 4. 20. A printing dyecomprising a xanthan composition according to claim
 6. 21. A printingdye comprising a xanthan composition according to claim
 9. 22. An oildrilling fluid comprising a xanthan composition according to claim 1.23. An oil drilling fluid comprising a xanthan composition according toclaim
 4. 24. An oil drilling fluid comprising a xanthan compositionaccording to claim
 6. 25. An oil drilling fluid comprising a xanthancomposition according to claim
 9. 26. A ceramic glaze comprising axanthan composition according to claim
 1. 27. A ceramic glaze comprisinga xanthan composition according to claim
 4. 28. A ceramic glazecomprising a xanthan composition according to claim
 6. 29. A ceramicglaze comprising a xanthan composition according to claim
 9. 30. Apharmaceutical composition comprising a xanthan composition according toclaim
 1. 31. A pharmaceutical composition comprising a xanthancomposition according to claim
 4. 32. A pharmaceutical compositioncomprising a xanthan composition according to claim
 6. 33. Apharmaceutical composition comprising a xanthan composition according toclaim
 9. 34. The pharmaceutical composition according to claim 30 whichis a controlled-release formulation.
 35. The pharmaceutical compositionaccording to claim 31 which is a controlled-release formulation.
 36. Thepharmaceutical composition according to claim 32 which is acontrolled-release formulation.
 37. The pharmaceutical compositionaccording to claim 33 which is a controlled-release formulation.
 38. Thepharmaceutical composition according to claim 34 which is acontrolled-release formulation.
 39. A method of producing a xanthanpolymer preparation having increased viscosity relative to that producedby a wild-type strain, comprising: selectively increasing the amount ofgene product of gumB and gumC but not of orfX and not of a gene selectedfrom the group consisting of gumD-gumG in a Xanthomonas campestrisculture, whereby a higher viscosity xanthan polymer preparation isproduced by the culture.
 40. The method of claim 39 wherein the step ofselectively increasing is performed by introducing into the Xanthomonascampestris one or more additional copies of gumB and gumC.
 41. Themethod of claim 39 wherein the step of selectively increasing isperformed by introducing into the Xanthomonas campestris one or moreadditional copies of gumB and gumC but not gumD-gumG.
 42. The method ofclaim 39 wherein the step of selectively increasing is performed byintroducing to the Xanthomonas campestris one or more additional copiesof gumB and gumC but not orfX and not gumD-gumG.
 43. The method of claim40 wherein the additional copies are on an extrachromosomal geneticelement.
 44. The method of claim 43 wherein the extrachromosomal geneticelement is a plasmid.
 45. The method of claim 44 wherein the plasmid isa broad host range plasmid.
 46. The method of claim 39 wherein theadditional copies are integrated in the genome of the Xanthomonascampestris.
 47. The method of claim 39 wherein the step of selectivelyincreasing is performed by inducing gumB and gumC expression using aninducible promoter and an inducing agent which increases expression fromthe inducible promoter.
 48. The method of claim 39 further comprisingthe step of recovering the higher viscosity xanthan polymer from thepreparation.
 49. The method of claim 39 further comprising the step ofprecipitating xanthan polymer from the higher viscosity xanthan polymerpreparation.
 50. A method of producing a xanthan polymer preparationhaving increased viscosity relative to that produced by a wild-typestrain, comprising: culturing a Xanthomonas campestris strain in aculture medium under conditions in which it produces a xanthan polymer,wherein the strain selectively produces more gene product of gumB andgumC but not of orfX and not of a gene selected from the groupconsisting of gumD-gumG relative to a wild-type strain.
 51. The methodof claim 50 wherein the strain has more than one copy of gumB and gumCper copy of gumD.
 52. The method of claim 50 wherein the strain has morethan one copy of gumB and gumC per copy of gumD-gumG.
 53. The method ofclaim 50 wherein the strain has more than one copy of gumB and gumC percopy of a gene selected from the group consisting of gumD-gumG.
 54. Themethod of claim 50 wherein the strain has more than one copy of gumB andgumC per copy of orfX.
 55. The method of claim 50 wherein the strain hasmore than one copy of gumB and gumC per copy of orfX and of gumD-gumG.56. The method of claim 50 wherein the strain carries one or moreplasmids which in aggregate carry at least one copy of gumB and gumC.57. The method of claim 50 further comprising the step of recovering ahigher viscosity xanthan polymer from the culture medium.
 58. The methodof claim 50 further comprising the step of precipitating xanthan polymerfrom the culture medium.
 59. An unpasteurized xanthan composition from acell which over-expresses gumB and gumC, wherein said composition has aseawater viscosity which is at least 10% greater than xanthan from acorresponding strain which does not over-express gumB and gumC.
 60. Thexanthan composition of claim 59 which has a seawater viscosity of DR>25when the seawater viscosity is measured in a solution of 41.95 g of seasalt per 1 liter deionized water and at a concentration of 0.86 gxanthan per 300 ml.
 61. The xanthan composition of claim 59 which has aseawater viscosity which is at least 15% greater than xanthan from acorresponding strain which does not overexpress gumB and gumC.
 62. Anoil drilling fluid comprising a xanthan composition according to claim59.
 63. An oil drilling fluid comprising a xanthan composition accordingto claim 61.